1. Field of the Invention
The present invention is in the field of medicinal chemistry. In particular, the invention is related to vaccines comprising novel combinations of saponin adjuvants, to pharmaceutical compositions and vaccines comprising these novel combinations, to methods of using these novel combinations to enhance the immune response of an individual to an antigen, and to the use of the novel combinations to increase the immunogenicity of vaccines.
2. Brief Description of the Background Art
Quillaja saponins are a mixture of triterpene glycosides extracted from the bark of the tree Quillaja saponaria. They have long been recognized as immune stimulators that can be used as vaccine adjuvants, (Campbell, J. B., and Peerbaye, Y. A., Res. Immunol. 143(5):526-530 (1992)), and a number of commercially available complex saponin extracts have been utilized as adjuvants. Crude saponins have been extensively employed as adjuvants in vaccines against foot and mouth disease, and in amplifying the protective immunity conferred by experimental vaccines against protozoal parasites such as Trypanosoma cruzi plasmodium and also the humoral response to sheep red blood cells (SRBC). (Bomford, Int. Arch. Allerg. Appl. Immun. 67:127 (1982)).
The first commercially available Quillaja saponin adjuvants were crude extracts which, because of their variability, were not desirable for use in veterinary practice or in pharmaceutical compositions for man. An early attempt to purify Quillaja saponin adjuvants was made by Dalsgaard, Archiv fuer die gesamte Virusforschung 44:243 (1974). Dalsgaard partially purified an aqueous extract of the saponin adjuvant material from Quillaja saponaria Molina. However, while Dalsgaard's preparation, "Quil-A," was a definite improvement over the previously available commercial saponins, it still exhibited considerable heterogeneity.
Subsequent analysis via high-pressure liquid chromatography showed that Quil A was in fact a heterogeneous mixture of structurally related compounds. (U.S. Pat. No. 5,057,540; Kersten, G. F. A. et al., Infect. Immun. 56:432-438 (1988); Kensil, C. R. et al., J. Immunol. 146:431-437 (1991); Kensil, C. R. et al., J. Am. Vet. Med. Assoc. 199:1423-1427 (1991)). However, not all of these saponins were active as adjuvants.
The four most predominant purified Quillaja saponins are QS-7, QS-17, QS-18, and QS-21 (alternatively identified as QA-7, QA-17, QA-18, and QA-21). These saponins have been purified by HPLC and low pressure silica chromatography and were found to be adjuvant active, although differing in biological activities such as hemolysis and toxicity in mice. In particular, QS-21 and QS-7 were found to be least toxic in mice. (Kensil, C. R. et al., J. Immunol. 146:431-437 (1991)).
Due to its potent adjuvant activity and low toxicity, QS-21 (commercially available as the "Stimulon.RTM." adjuvant) has been identified as a useful immunological adjuvant. (Kensil, C. R. et al., "Structural and Immunological Characterization of the Vaccine Adjuvant QS-21," in Vaccine Design: The Subunit and Adjuvant Approach, Powell, M. F. and Newman, M. J. eds., Plenum Press, New York (1995)). QS-21 is a complex triterpene glycoside of quillaic acid. QS-21 is glycosylated at triterpene carbon 3, triterpene carbon 28, and carbon 5 of the second fatty acyl unit in a fatty acid domain.
More recently, QS-21 was further purified using hydrophilic interaction chromatography (HILIC) and resolved into two peaks, QS-21-V1 and QS-21-V2, which have been shown to be chemically different compounds. In C57BL/6 mice immunized with vaccines consisting of ovalbumin and either QS-21, QS-1-V1, or QS-21-V2, both of the individual components QS-21-V1 and QS-21-V2 are comparable in adjuvant effect to the original QS-21 peak (containing a mixture of 3:2 QS-21-V1 and QS-21-V2) for boosting the IgG subclasses IgG1, IgG2b, and IgG2 as well as the total IgG titer. (Co-pending U.S. patent application No. 07/906,880, U.S. Pat. No. 5,583,112, the entire contents of which is hereby incorporated by reference).
Quillaja saponins are structurally distinct from the saponins derived from other plant species. Two structural features that distinguish Quillaja saponaria saponins from those of other plant species are a fatty acid domain and a triterpene aldehyde at carbon 4 of the triterpene. (Kensil, C. R. et al., "Structural and Immunological Characterization of the Vaccine Adjuvant QS-21," in Vaccine Design: The Subunit and Adjuvant Approach, Powell, M. F. and Newman, M. J. eds., Plenum Press, New York (1995)). Modifications to the aldehyde on the triterpene indicate that this functional group may be involved in the adjuvant mechanism (Soltysik, S. et al., Vaccine 13(15):1403-1410 (1995)).
Quillaja saponins, particularly QS-7, QS-17, QS-18, and QS-21, have been found to be excellent stimulators of antibody response to soluble T-dependent protein antigens, "subunit antigens", which are poorly immunogenic and require a potent adjuvant for maximization of immune responses. Examples of purified subunit antigens for which saponin adjuvants will augment the IgG response in mice include keyhole limpet hemocyanin (KLH), HIV-1 gp120 (Bomford, R. et al, AIDS Res. Hum. Retroviruses 8:1765 (1992)), and influenza nucleoprotein (Brett, S. et al., Immunology 80:306 (1993)). QS-7, QS-17, QS-18 and QS-21 have also been shown to stimulate potent antibody responses in mice to the antigens bovine serum albumin and cytochrome b.sub.5 (Kensil, C. R. et al., J. Immunol. 146:431 (1991)). The level of antibody response induced by these purified saponins was comparable to other commonly used adjuvants, e.g., complete Freund's adjuvant, and superior to aluminum hydroxide.
QS-21 has also been shown to enhance antibody responses to T-independent antigens, including unconjugated bacterial polysaccharides (White, A. C. et al., "A purified saponin acts as an adjuvant for a T-independent antigen, in: Immunobiology of Proteins and Peptides, Vol. VI (M. Z. Atassi, ed.), Plenum Press, New York, pp. 207-210 (1991)). The immunogenicity of the vaccine was further increased by conjugating diphtheria toxoid to the polysaccharide. QS21 enhanced the antibody response to the polysaccharide as well as the carrier, including IgG2a, IgG2b, and IgG3 responses. (Coughlin, R. T. et al., Vaccine 13(1):17-21 (1995)).
The ability of adjuvants to modulate the isotype distribution and IgG subclass distribution of antibody response to an antigen through the promotion of Ig subclass switching has important implications for immunity to many bacterial and viral vaccines. QS-7, QS-17, QS-18, and QS-21 stimulate IgG2a response to cytochrome b5 after administration with saponin doses of 20 .mu.g (Kensil, C. R. et al., J. Immunol 146:431 (1991)). In this regard, QS-21 shifts predominant IgG1 responses to a profile that includes significant IgG2b and IgG2a responses. For example, QS-21 has been shown to stimulate antigen-specific IgG2a to a number of antigens, including Borrelia burgdornferi outer surface proteins OspA and OspB (Ma, J. et al., Vaccine 12(10):925 (1994)), feline leukemia virus (FeLV), envelope gp70 (Kensil, C. R. et al., J. Am. Vet. Med. Assoc. 10:1423 (1991)), human cytomegalovirus (HCMV) envelope protein gB (Britt, W. et al., J. Infect. Dis. 171:18 (1995)), respiratory synctial virus (RSV) purified fusion protein (Hancock, G. E. et al., Vaccine 13(4):391 (1995)), and tetanus toxoid (Coughlin, R. T. et al., Vaccine 13(1):17 (1995)). QS-21 has also been shown to induce boostable antibody responses. (Britt et al., J. Infect. Dis. 171:18-25 (1995); Helling et al., Cancer Res. 55:2783-2788 (1995)).
The ability of the QS-21 adjuvant to induce class I major histocompatibility complex (MHC) antigen-restricted cytotoxic T-lymphocyte responses (CTL) after immunization with soluble proteins is a characteristic of saponin adjuvants. A number of studies have shown the ability of QS-21 to induce potent cytotoxic T-lymphocyte (CTL) responses to various antigens, including ovalbumin (Wu, J.-Y. et al., Cell. Immunol. 154:394-406 (1994); Newman, M. J. et al., J. Immunol. 148(8):2357-2362 (1992)), recombinant HIV-1 gp160 protein (Wu, J.-Y. et al., J. Immunol. 148:1519 (1992)), and subunit SIV.sub.mac251 gag and env (Newman, M. J. et al., AIDS Res. Hum. Retroviruses 10(7):853 (1994)).
Most of the saponin adjuvant studies have been carried out in mice. However, the adjuvant activity of saponins is not limited to mice; it has also been demonstrated in guinea pigs, rabbits, pigs, sheep, cattle, and nonhuman primates. An adjuvant effect from QS-21 has been observed in cats, guinea pigs, dogs, nonhuman primates, and humans. (Kensil, C. R. et al., "Structural and Immunological Characterization of the Vaccine Adjuvant QS-21," in Vaccine Design: The Subunit and Adjuvant Approach, Powell, M. F. and Newman, M. J. eds., Plenuim Press, New York (1995)).
Phase 1 human trials of QS-21 with GM2 ganglioside-keyhole limpet haemocyanin conjugate vaccine have been conducted in patients with malignant melanoma (Livingston, P. O. et al., Vaccine 12:1275-1280 (1994). Increased immunogenicity after administration with QS-21 adjuvant was observed (Helling, F. et al., Cancer Res. 55:2783-2788 (1995)). In another set of clinical trials, QS-21 was found to be a potent immunological adjuvant that significantly increased the serological response of melanoma patients to the murine antiidiotype antibody MELIMMUNE-1 (Livingston, P. O. et al., Vaccine Res. 4(2):87 (1995).
A number of studies discuss the use of Quillaja saponins, particularly QS-21, in conjunction with other adjuvants. For example, QS-21 was shown to be an effective co-adjuvant with aluminum hydroxide (alum)--absorbed antigens. (Ma, J.-Y. et al., Vaccine 12(10):925-933 (1994); Newman, J. et al., J. Immunol. 148(8):2357-2362 (1992); Kensil, C. R. et al., "Structural and Immunological Characterization of the Vaccine Adjuvant QS-21," in Vaccine Design: The Subunit and Adjuvant Approach, Powell, M. F. and Newman, M. J. eds., Plenum Press, New York (1995); Kensil et al., J. Am. Vet. Med. Assoc. 199:1423-1427 (1991); Wu, J.-Y. et al., J. Immunol. 148:1519-1525 (1992); Kensil et al., Vaccine Res. 2:273-281 (1993)). Moreover, the use of mixtures of two or more saponin adjuvants is discussed in U.S. Pat. No. 5,057,540, and currently co-pending U.S. patent application No. 07/906,880 (now U.S. Pat. No. 5,583,112) (The entire contents of both of these documents are hereby incorporated by reference.)
The immune adjuvant effect of saponins is dependent upon dose. Depending upon the antigen and the species, a minimum dose level of QS-21 is required for optimum response. (Kensil, C. R. et al., J. Immunol. (1991); Kensil, C. R. et al., Vaccine Res. (1993); Newman et al., J. Immunol. (1992); Livingston, et al., Vaccine (1994). Below this minimum dose, the immune adjuvant effect is suboptimal (either low level or absent). QS-7 also has a dose response curve. (Kensil, C. R. et al., J. Immunol. (1991)).
Until now, however, the identification of combinations of two or more Quillaja saponins in suboptinal doses to produce a synergistic adjuvant effect was unknown in the art.